The Project (Transcription-Coupled and Replication-Associated Excision Repair) focuses on mechanisms coupling DNA excision repair machinery with transcription and replication. Both[unreadable] Nucleotide Excision Repair (NER) and Base Excision Repair (BER) are highly coordinated by interactions[unreadable] between proteins in the pathway. Moreover, they are preferentially targeted by specialized transcriptioncoupled[unreadable] repair (TCR) machinery to lesions that affect transcription elongation or by replication-associated[unreadable] repair (RAR) to lesions near the replication fork or in recently replicated DNA. We hypothesize that these[unreadable] interactions and their effects on function are regulated through unstructured flexible regions that undergo[unreadable] disorder-to-order transformations upon complex formation and/or post-translational modifications. We will[unreadable] test this overall hypothesis and specific hypotheses in five Aims by collaborative studies to characterize,[unreadable] validate, and map interactions, identify damage-induced modifications, observe effects of complexes on DNA[unreadable] structure by scanning force microscopy (SFM), and visualize subunits and complexes by electron[unreadable] microscopy (EM), small angle X-ray scattering (SAXS), and protein crystallography (PX). Aim 1 will[unreadable] structurally characterize early steps of TCR: recognition by XPG and CSB of RNA Polymerase II (RNAPII)[unreadable] stalled at a lesion, and remodeling of RNAPII by TFIIH to allow access to the lesion. SFM and EM studies[unreadable] will test the hypothesis that these occur by ordered conformational changes. Aim 2 will structurally[unreadable] characterize CSB and reinvestigate its causal role in CS by determining whether mutant CSB interferes with[unreadable] responses to oxidative DNA damage through non-productive interactions with other proteins in the pathway.[unreadable] Aim 3 will investigate the identified interactions that couple BER and NER to transcription through (a) SAXS[unreadable] and PX studies of XPG protein and its domains and complexes, (b) analysis of interactions of NEIL2 with[unreadable] RNAPII, XPG and CSB, and (c) characterization of the effect of post-translational modifications on XPG and[unreadable] NEIL2 interactions. Aim 4 will characterize the structural basis for BER pathway coordination by interactions[unreadable] of NEIL1 and NEIL2 glycosylases with downstream BER proteins and test the hypothesis that BER pathway[unreadable] progression results in progressive DNA bending. Aim 5 will investigate molecular mechanisms of RAR by[unreadable] determining the structure of the checkpoint sliding clamp -- the 9-1-1 complex - and by characterizing[unreadable] interactions of the MYH and NEIL1 glycosylases with PCNA and 9-1-1. The anticipated outcome is a[unreadable] molecular understanding of cancer predispositions and developmental disorders that arise from defects in[unreadable] the coordination of excision repair with transcription and replication. Collaborations of Project 2 within SBDR[unreadable] and with the UCSF Comprehensive Cancer Center will relate results of these studies to genome integrity and[unreadable] cancer etiology as well as to development of promising molecular targets for cancer drug discovery.